Tumor invasion and metastasis is characterized by a series of processes that involve the cancer cells and the host extra cellular matrix. Basement membranes are specialized extra cellular structures that play an important role in organizing the cells that rest on them. Metastases of tumor cells involve interaction of the cells with the components of extracellular matrix (ECM). Laminin is a major component of the basement membrane which promotes cellular attachment, proliferation, growth, differentiation and migration (Kleinman H K et al. J Cell Biochem 1985, 217:317-25. Martin G et al, Annual Rev Cell Biol 1987, 3:57-85. Beck K et al, FASEB J 1990, 4:148-60). In vitro and in vivo models have shown tumor cell binding to laminin to be associated with cancer invasion, migration and the cability to metastasize (Terranova V P et al, Cancer Res 1982, 42:2265-2269. Varani J et al, Am J pathol 1983, 111:27-34. Barsky S H et al, J Clin Invest 1984, 74:843-848. Malinoff H L, Int J Cancer 1984, 33:651-655. Kanemoto K et al, Proc Natl Acad Sci USA 1990, 87:2279-2283). The 67 kD laminin receptor (67LR), is a non-integrin high affinity laminin-binding protein whose expression is significantly increased in cancer cells and interacts with laminin among other cell surface proteins (Malinoff H L et al. Int J Cancer 1984, 33:651-655. Rao C N et al. Bicohem Biophys Res Commun 1983, 111:804-808. Terranova V P et al. Proc Natl Acad Sci USA 1983, 80:444-448. Malinoff H et al. J Cell Biol 1983, 96:1475-1480. Ruyman R B et al. J Cell Biol 1988, 107:1863-1871. Albelda S M et al. FASEB J 1990, 4:2868-2880. Hail D E et al. J Cell Biol 1990, 110:2175-2184). It has been shown that expression of the 67LR is increased in cancers such as breast, colon, and gastric carcinomas compared to normal tissues (Cioce V et al. J Natl Cancer Inst 1991, 83:29-36. Castronovo V et al. Am J Pathol 1990, 137:1373-1381. D'Errico A et al. Mod Pathol 1991, 4:239-246).
The 67 kD laminin receptor (67LR; Table 1) was originally isolated from cell membranes of murine melanoma (Rao et al, 1983. Biochem Biophys Res. Commun. 111:804-808), fibrosarcoma cells (Malinoff & Wicha, 1983. J Cell Biol 96: 1475-1479) and normal bovine muscle cells (Lesot et al, 1983. EMBO J. 2: 861). Since then it has been detected in a number of species and is present in a broad range of human tissues (for review see Barsoum Rohrer and Coggin 2000. Cell Mol Biol Lett. 5: 207-230; Menard et al, 1998 J. Cell. Biochem. 67:155-165; Mecham 1991 Annu Rev Cell Biol 7:71-91).
TABLE 1Synonyms for Laminin receptor 1 and precursorProteinNamesLaminin receptor 1Laminin receptor 1 (LamR1/LAMR1/LR1)Laminin receptor (Laminin-R/LN-R/LR)Laminin binding receptor (LAMBR/LBR)67 kD Laminin receptor (67 kD LR/67LR)Colon carcinoma laminin binding proteinLaminin receptor 1Laminin receptor precursor (LRP);Precursor37 kD Laminin receptor precursor (37LRP)37 kD Laminin binding protein (37LBP)Immature Laminin receptor protein (iLRP):32 kD Laminin binding protein (LBP 32)
Human cDNA for the 67LR was originally isolated from a malignant colon carcinoma (hence the protein is also known as the Colon Carcinoma Laminin Binding Protein; Yow et al, 1988. 85: 6394-6398) and is smaller than first anticipated. The cDNA predicted sequence encodes for a 295 amino acid protein with a calculated mass of 32 kD. However, it normally runs at 37-44 kD on an SDS-PAGE gel, which may be due to the reduced electrophoretic mobility of an acidic protein (calculated pI 4.83). A small number (16) of hydrophobic amino acids are present towards the N-terminus (Castronovo, Taraboletti & Sobel, 1991. J. Biol. Chem. 266: 20440-20446) that may span the cell membrane. The 37 kD protein and the 67 kD protein were shown to be antigenically related (Rao et al, 1989; Biochemistry 28: 7476-7486) while pulse chase experiments performed on melanoma cells indicated that the 37 kD protein generated was chased into the 67 kD product (Castronovo et al, 1991. Biochem. Biophys. Res. Commun. 177:177-183). These results suggested that there was a direct precursor-product relationship between the two proteins. Hence, the 37 kD protein is referred to as the 37 kD laminin receptor precursor (37LRP; Table 1). The pulse chase experiments did not reveal the presence of any intermediate forms between the precursor and the final 67LR (Castronovo et al, 1991), although a 50 kD degradation product was detected. The 37 kD polypeptide may have a multifunctional role in the cytoplasm and the membrane and may be the ligand binding component of the 67LR (Elias Campo et al. Am J pathol 1992, 141:No. 5 1073-1083). In cancer cells it has been shown that antibodies to the 67 kD protein bind to both the cell surface and cytoplasm (Wever U M et al. Cancer Res 1987, 47:5691-5698).
It is not fully understood how the final receptor is made by cells to achieve the 67 kD receptor, but acylation by the fatty acids palmitate, oleate and stearate may be involved (Landowski, Dratz, & Starkey, 1995. 34: 11276-87; Buto et al, 1998 J. Cell Biochem. 69: 244-251). Extensive glycosylation is not involved. The predicted cDNA sequence does not contain a consensus sequence site for N-linked glycosylation and despite the presence of serine and threonine residues, there is no evidence of O-linked glycosyl groups (Castronovo et al, 1991. Biochem. Biophys. Res. Commun. 177:177-183; Landowski, Dratz,& Starkey, 1995. 34: 11276-87). However, Castronovo (Castronovo, 1993 Invasion Metastasis 13:1-30) suggested that the 67LR expresses epitopes that cross-react with β-galactosidase-binding lectins. The 67LR may comprise of a dimer of the precursor polypeptide linked by lipids (Landowski, Dratz & Starkey, 1998). It has also been suggested that heterodimerisation may occur with a lectin-like protein or galectin-3 (Castronovo et al, 1991; Buto et al, 1998). Anti-galectin-3 antibodies recognised not only galectin-3 but also the 67LR (Buto et al, 1998). The final structure of the receptor remains to be elucidated.
The 67LR, when shed from the surface of cells in culture, retains its capacity to bind to laminin (Karpatova et al, 1996. J. Cell. Biochem. 60:226-503). It remains uncertain how the 67LR is attached to the cell membrane. Although the receptor has 16 hydrophobic amino acids towards its' N-terminal end, it is possible that it interacts with associated molecules rather than existing as an integral membrane protein. However, it has been established that the amino-terminal of the polypeptide is inaccessible in non-permeabilised cells suggesting that indeed this region interacts with other molecules (Castronovo et al, 1991. J. Biol. Chem. 30 20440-20446; Wewer et al, 1987 Cancer Res. 47:5691-5698).
It has also been suggested that accessory factors may be associated with the 67LR, or that it acts as an accessory molecule itself. Such properties may assist in transport to the cell surface and/or laminin binding. It has been noted that co-expression of 67LR and α6β1 in small cell lung cancer cell lines directly correlated with cell adhesion to laminin (Pellergrine et al, 1994. Int J Cancer Suppl 8: 116-120). When human melanoma cells were treated with laminin both 67LR and α6β1 co-translocated to the plasma membrane (Romanov et al, 1994. Cell Adhes Commun. 2:201-209). 67LR associated with α6β1 mediated high-avidity adherence of a population of human memory T cells to laminin (Clanfield and Khakoo, 1999. J. Immunol. 163: 3430-3440). Ardini et al, 1997 noted that the 67LR and α6β4 not only co-localised but were co-regulated, via physical interactions between the 67LR and α6 subunit (Ardini et al, 1997; J. Biol. Chem. 272:2342-2345). However, in ovarian carcinomas expression of 37LRP mRNA and protein is independent of the α6 subunit (Givant-Horwitz, 2003 Clin. Exp. Metastasis 20:599-609; Skubitz et al, 1996. Am J pathol 148:1445-1461). Together these results suggest that the 67LR may associate with laminin-specific integrins (in particular the α6 subunit) in the cytoplasm, arriving at the cell membrane as a complex where both receptors participate in the recognition of laminin and determining whether the interaction is one of high or low affinity (Landowski, Dratz, Starkey, 1995).
The active human 37LRP gene maps to 3p21.3, a chromosomal locus that is frequently involved in genetic alterations associated with cancers (Jackers et al, 1996. Oncogene 13: 495-503). The active gene contains seven exons and six introns (Jackers et al 1996. Oncogene 13: 495-503; avian gene Clausse et al, 1996 DNA Cell Biol 15: 1009-1023). It does not contain a classical TATA box but there may be multiple transcription start sites. There are four Sp1 sites present in the promoter region, six Sp1 sites in intron 1 and two Alu sequences in intron 3 that may affect alternative splicing. Intron 4 contains the sequence for the small nuclear RNA E2 (Jackers et al 1996. Oncogene 13: 495-503). At least 26 copies of the gene are present in the human genome, all demonstrating high homology with the functional gene (Jackers et al, 1996. Biochem Biophys Acta. 1305:98-104). Nineteen of these copies were analysed and were shown to be processed pseudogenes giving rise to dysfunctional transcripts. It is thought that these pseudogenes have most probably been generated by retropositional events (Jackers et al, 1996. Biochem Biophys Acta. 1305:98-104). The cDNA is highly conserved throughout evolution with at least 98.3% homology among mouse, bovine and human sequences while the rat and human sequences share 99% homology (For review see Menard et al, 1997. J Cell Biochem 67:155-165).
The 37LRP gene appears to give rise to a number of functional proteins other than the 37LRP. The 37LRP protein shares 99% homology with the p40 ribosome-associated protein (p40 polypeptide; p40; ribosomal protein SA; RPSA) involved in the translational machinery (Makrides et al, 1988. Nucleic Acid Res. 16: 2349; Tohgo et al, 1994. FEBS Lett. 340: 133-138; Rosenthal & Wordeman 1995. J. Cell Sci. 108: 245-256). A positional marker in the development of the embryonic eye is also encoded for by a gene identical to the 37LRP cDNA (Rabacchi et al, 1990. Development 109: 521-531; McCafferey, Neve and Drager, 1990. PNAS 87: 8570-8574).
The oncofetal antigen (OFA; 37-44 kD) is an immunogenic glycoprotein expressed in rodent and human tumors and early foetuses. The murine 37LRP shares up to 99.5% identity with OFA (Coggin, Barsoum, Rohrer 1999. Anticancer Research 19: 5535-5542). It has been referred to as the auto-immunogenic homologue of 37LRP. OFA has been shown to stimulate T and B lymphocytes in both mice and humans, and play an immunogenic role in cancers, in particular in renal cancers (Zelle-Rieser et al, 2001. J. Urol. 165:1705-9; Holt et al, 2002. Clin. Cancer Res. 8:3369-3376; Rohrer et al, 1992; J. Natl. Cancer Inst. (Bethesda) 84: 602-609; Rohrer et al, 1994. J. Immunol. 155: 755-764; Rohrer et al, 1995. J. Immunol. 155:5719-5727; Rohrer et al, 2001. Mod. Aspects Immunibiol. 1: 191-195; Rohrer et al, 1999. J. Immunol 162: 6880-6892).
There is some evidence to suggest that isoforms or homologues of the 37LRP and 67LR may exist. A 55 kD protein has been identified in human and bovine endothelial cells sharing identity with 37LRP (Ireland et al, 1998. Clin. Exp. Immunol. 112:255-261), and a number of isoforms have been found in murine brain tissue (Simoneau et al, 2003. Biol. Chem. 384:243-246). These proteins may arise from the 37LRP being post-translationally modified in various ways and/or interacting with other molecules, or may arise from other highly homologous genes.
Over-expression and abnormal surface distribution of the 67LR has been demonstrated in a broad range of tumors, detected by various technologies at the mRNA and protein levels (For review see Menard et al 1998; Barsoum et al, 2000). Change in levels of 37LRP and/or 67LR have been shown to affect tumor biology in terms of disease progression, invasiveness, metastasis, aggressiveness and prognosis.
The over-expression of the 67LR has been associated with the receptor playing a role in tumor progression, although the stage of progression may be dependent on the tumor type (Campo et al, 1992. Am J Pathol 41:1073-83; Demeter et al, 1992 Cancer Res. 52:1561-1567; Martignone et al, 1992. Clin. Exp. Metastasis 10:379-386: breast cancer; Vasso et al, 1993; Cancer 15: 455-461: melanoma; Boukerche et al, 2004. Gene 343:191-201: melanoma; Waltregny et al 1997. J. Natl. Caner Inst 89:1224-1227). An increase in 37LRP mRNA in frozen colorectal tissues could be seen in adenocarcinomas compared with adenomas, whereas levels were constant between normal and adenoma tissues. These results suggest that expression of 37LRP or 67LR correlated with a late event in disease progression from adenoma to adenocarcinoma/Dukes C carcinoma (Campo et al, 1992. Am J Pathol 41:1073-83.). In contrast, 37LRP mRNA increased in adenomatous cervical lesions suggesting an early event in disease progression (Demeter et al, 1992 Cancer Res. 52:1561-1567). 67LR has also been implicated as a lineage-associated antigen in monocytic acute myeloid leukaemia (AML; Montouri et al, 1999. Clin. Cancer Res. 5:1465-1472).
Other studies have shown that the 67LR may play a role in invasiveness and metastasis, implying that it plays a significant role in the acquisition of a metastatic phenotype in various types of tumors (Wewer et al, 1987. Cancer Res 47: 5691-8; Castronovo & Sobel 1990. Biochem Biophys Res Commun 68: 1110-1117; Cioce et al, 1991. J Natl Cancer Inst 83: 29-36; Sobel, 1993 Semin. Cancer Biol. 4: 311-317; Castronovo Invasion Metastasis 1993 13:1-30; You et al, 1988. PNAS 85: 6394-6398; Pelosi et al, 1997. J. Pathol. 183:62-69; Boukerche et al, 2004. Gene 343:191-201). For example, levels of mRNA have been shown to increase in human colon cell lines and tissues with greater malignant potential (Kondah et al, 1992. Cancer Res 52: 791-796). Inhibition of metastasis of a human fibrosarcoma cell line occurred when cells were pre-treated with an IgG fraction (P4G) of sera from rabbits immunised with a 37LRP-GST fusion protein (345 bp cDNA; 13 kD; Narumi et al, 1999. Jpn J. Cancer Res. 90: 425-431). The sera also reduced cell attachment to laminin in a dose-dependent manner. Antisense RNA of 37LRP also inhibited invasiveness of a poorly differentiated human colon carcinoma cell line in vitro (Mafune and Ravikumar, 1992. J. Surg. Res. 52:340-346).
The increase in 67LR expression during metastasis is often paralleled by the decrease in expression of another non-integrin laminin binding protein, galectin-3 (van den Brule et al, 1994. Eur. J. Cancer 32A:1598-1602; Xu et al, 1995. Am. J. Pathol. 147:815-822; Castronovo et al, 1995. J. Pathol. 179:43-48; Lotz et al, 1993. PNAS 90: 3466-3470). These results suggest that these two laminin receptors are inversely regulated and this may account for changes in laminin-binding affinity depending on which receptor is being used. In contrast, a direct correlation between increased expression of galectin-3 and the malignancy of colon carcinomas has been observed (Schoeppner et al, 1995 Cancer 75:2818-2826).
67LR expression may also be a marker for aggressiveness of a tumor since increased expression tends to be associated with proliferation and marked tumor growth. Increased levels of 37LRP mRNA were detected in human lung cancer tissues (Satoh et al, 1992. Biochem. Biophys. Res. Commun 182: 746-752) and pancreatic endocrine tumors (Pelosi et al, 1997. J. Pathol. 183: 62-69) that were rapidly proliferating. In cervical neoplasms associated with human papillomavirus, increased levels of 37LRP mRNA were correlated with proliferative properties of the cells rather than with the invasive properties of the cells (Demeter et al, 1992). Introduction of antisense 37LRP RNA into murine lung cancer cell line T11 prolonged their doubling time (Satoh et al, 1999. Br. J. Cancer 80:1115-1122). These cells also displayed weaker interactions with laminin and survival time in mice subcutaneously inoculated with cells treated with antisense RNA was prolonged. The 67LR may also play a role in tumour aggressivenss since it may enhance proteolytic cleavage of laminin-1, therefore assisting in the degradation rate of the basement membrane (Ardini et al, 2002. Cancer Res. 62: 1321-1325).
Over-expression of the 37LRP and/or 67LR may also be associated with poor prognosis in several types of tumors (for review see Barsoum Rohrer and Coggin 2000. Cell Mol Biol Lett. 5: 207-230; Menard et al, 1998 J. Cell. Biochem. 67:155-165; Menard, Tagliabue and Colnaghi, 1998. Breast Cancer Res. Treatment 52: 137-145). Prognosis is unfavourable in breast carcinomas that are also producing laminin (Martigone et al 1993. J. Natl. Cancer Inst. 85: 379-386; Pellegrini et al, 1985 Breast Cancer Res Treat 35: 195-199). In human lymphomas, 67LR was detectable on the surface of CD30+ anaplastic large cell lymphomas and in small subsets of high-grade B-cell non-Hodgkin's or Hodgkin's lymphomas (Carbone et al, 1995. Hum. Pathol. 2: 541-546).
Recently, the 67LR has been implicated in biological processes other than tumor biology. The receptor was found to be up-regulated by cytokines, inflammatory reagents, interactions with extracellular matrix proteins including laminin and steroids (for review see Menard et al, 1998), suggesting that the 37LRP and 67LR may be regulated under normal conditions. The receptor may play a role in lymphocyte chemotaxis, adhesion and homing and/or in host defence mechanisms. The 67LR has been found on the surface of a population (10-15%) of human activated memory peripheral blood T cells (both CD4+ and CD8+ single positives). It has also been shown to be up-regulated in response to neuropeptides (Chen et al, 2002 Nat. Med. 8:1421-1426). A study by Ferrarini et al, 1996 supports an immunological role for the receptor since γδ+ lymphocytes localised in lung tumor sites were capable of killing lung cancer cells, mediated by interactions with 67LR (Ferrarini et al, 1996. J. Natl. Cancer Inst. 88:436-441). The killing was shown to be independent of natural killer (NK) cells, lymphokine-activated (LAK) cells and the T cell receptor (TCR) whereas laminin could provide a co-stimulatory signal.
The 67LR may also affect the growth, migration and trafficking of other cell types. 67LR interacts with the a (αGMR) and β (βGMR) subunits of the GM-CSF receptor (Chen et al, 2003. PNAS 100: 14000-14005) and inhibits the formation of the GM-CSF receptor complex. GM-CSF regulates the growth, differentiation and maturation of myeloid precursor cells and enhances the function of mature neutrophils, eosinophils and mononuclear phagocytes. 67LR may inhibit these activities by preventing GM-CSF complex formation. Secretory and endocytic roles for the 67LR have also been implied since it has been found in the brush border and in Paneth cell secretory granules (Shmakov et al, 2000. J. Pathol. 191:318-322).
The precise way in which 67LR interacts with laminin remains undefined. Two peptide domains have been identified from the 67LR as possible laminin binding sites. One of these, Peptide G, a synthetic peptide derived from the sequence of 37LRP, contains the palindromic sequence LMWWML (SEQ ID NO:1). It was shown to bind laminin, to inhibit binding of tumor cells to endothelial cells, and to increase the metastases of human melanoma cells in nude mice (Castronovo et al, 1991 J Biol. Chem. 266:20440-20446; Castronovo, Taraboletti and Sobel 1991. Cancer Res. 51:5672-5678; Taraboletti et al, 1993. J. Natl. Cancer. Inst. 85:235-240). It was discovered that peptide G increases and stabilizes the binding of laminin on tumor cells (Magnifico et al, 1996. J. Biol. Chem. 271:31179-31184). The second possible laminin binding domain was predicted from the hydrophobicity of the C-terminal sequence of the 37LRP (a.a. 205-229; Landowski, Uthayakumar, Starkey, 1995. Clin. Exp. Metastasis 13:357-372). It is also possible a lectin domain of the 67LR interacts with laminin since laminin recognition of the receptor is dependent on lactose (Castronovo et al, 1991. Biochem. Biophys. Res. Commun. 177:177-183).
The 67LR may bind to laminin residues YIGSR (SEQ ID NO:2) (a.a.929-933; β1 chain; Massi, Rao and Hubbell, 1993. J. Biol. Chem. 268:8063-8059; Landowski, Uthayakumar, Starkey, 1995. 13:357-72), IKVAV (SEQ ID NO:3) (a.a.2091-2108; α chain; Kibbey et al, 1993. PNAS 90:10150-10153) and LGTIPG (SEQ ID NO:4) (a.a.442-446; β1 chain; Mecham et al 1989 J. Biol. Chem. 264:16652-16657). The 67LR may also bind to the carbohydrate components of laminin, in particular poly(lactosamino) structures, Gal (β1, 3)Gal linkages and terminal non-reducing β-galactosyl residues (Mecham, 1991 Annu. Rev. Cell. Biol. 7:71-91). Binding to the residues YIGSR (SEQ ID No:2) inhibits metastasis (Iwamoto et al, 1996. Br. J. Cancer 73:589-595), while metastasis is stimulated by IKVAV (SEQ ID NQ:3) interactions (Bresalier et al, 1995. Cancer Res. 55:2476-2480). Since the discovery of the first laminin receptor, 67LR, in 1983 at least 14 other laminin receptors have been described (Mecham 1991) and may utilize the same binding sites on laminin.
The 67LR also interacts with other molecules in addition to laminin. These include elastin (Grosso et al, 1991. Biochemistry 30: 3346-3350), fibronectin (FN), type IV collagen (Narasimhan et al, 1994. PNAS 91:7440-7444; Iwabuchi et al, 1996 Blood 87: 365-372) and heparin (Guo et al, 1992. PNAS 89: 3040-3044). Other studies have also shown that the 67LR serves as a receptor for sindbis virus (Wang et al, 1992 J. Virol. 66:4992-5001), while the 37LRP allows uptake of prion proteins (Rieger et al, 1997. Nat. Med. 3:1383-1387).
Liver Cancer
The most common primary malignant tumor of the liver is hepatoceullar carcinoma (HCC). The incidence of HCC is increased in populations who are at high risk for Hepatitis B and C. Patients already suffering from chronic hepatitis, cirrhosis, hemochromatosis, and the two congenital hepatic disorders, alpha-1-antitrypsin deficiency and tyrosinema, are also at higher risk of developing HCC. Certain toxins and chemicals may also cause primary liver cancer, including aflatoxin, a product from mould found in improperly stored peanuts in Africa. If HCC is successfully removed by resection, recurrence and metastasis are likely. A number of studies have revealed various prognostic markers for primary liver cancer and metastatic recurrence (For review see Qin and Tang, 2004. J Cancer Res Clin Oncol 130: 497-513).
The association of liver diseases with HCC suggests architectural changes may increase the chance of developing primary liver cancer. Under normal conditions hepatocytes are characterised by the absence of a basement membrane (Schaffner and Popper 1963 Gastroenterology 44:230-242). Extracellular matrix proteins, including laminin, can be produced as cirrhosis develops and are deposited around sinusoids, forming a structured basement membrane. Laminin 5 was found to be present in primary HCC nodules but not in normal or peri-tumoral cirrhotic tissues (Giannelli et al, 2003. Clin. Can. Research. 9:3684-3691). Laminin has also been shown to induce the expression of cytokeratin 19 suggesting that laminin deposits cause abnormal expression of other proteins (Su et al, 2003. World J Gastroenterol : 921-929). It may be that the expression of laminin increases expression of any of its receptors.
The 67LR was found to be expressed in hepatocytes in 1990 (Clement et al, 1990. J. Cell Biol. 110:185-192), although it was not the only laminin receptor present. An increase in the number of 67LR positive cells was observed in neoplastic regions compared to adjacent parenchyma in liver samples taken from patients with HCC and cirrhosis (Grigioni et al (1991, Am J Pathol 138:647-654). Another study (Ozaki et al, 1998. Gut 43: 837-842) detected weak 37LRP mRNA expression in normal liver tissues. mRNA levels increased in non-cancerous liver tissue with chronic liver disease and were elevated further in tumor regions. 37LRP translation and expressed protein were not determined in this study (Ozaki et al, 1998. Gut 43: 837-842). Increased biosynthesis of the 67LR was observed in the metastatic HCC tissues with a direct correlation between increases in RNA and protein (Zheng et al, 1997. J Tongji Medical University. 17:200-202). L-02 normal hepatic cells and the cancer cell lines HepG2 and SMMC-7721 showed varied patterns of 37LRP mRNA and 67LR expression that did not correlate with the tumor state of the cell line (Zheng et al, 2002. Chinese J Cancer 22: 248-252). However, the carcinoma cell line SMMC-7721 may express higher laminin binding affinity than the other cell lines although this cannot be attributed to the 67LR alone since whole cells were used for the binding studies. However, a proteomics study revealed that 67LR is up-regulated in the highly metastatic cell line MHCC97-H, compared to the low metastatic counterpart MHCC97-L (Li et al, 2001. World J Gastroenterol 7: 630-636; Ding et al, 2004. Proteomics 4; 982-994). Whether 67LR plays a direct role in HCC metastasis remains to be determined.